Hyaluronic acid is a linear macromolecular polysaccharide consisting of alternately bonded β-D-N-acetylglucoamine and β-D-glucuronic acid. Hyaluronic acid is found not only in connective tissues of mammals but also in cockscombs and the capsules of Streptococci. Hyaluronic acid is obtainable not only by extraction from cockscombs and umbilical cords, but also as purified products from the culture broth of streptococci. 
Natural hyaluronic acid is polydisperse in respect of molecular weight and is known to show excellent biocompatibility even when implanted or injected into the body by virtue of the absence of species and organ specificity. However, because of the relatively short in vivo residence time of hyaluronic acid solution in biological application, improvement of the persistency of hyaluronic acid by chemical crosslinking with various chemical modifiers has been attempted to broaden its use for medical materials.
(I) Concerning the joints, synovial fluid supplies nutrition to the articular cartilage and has incomparable functions as a lubricant and a shock absorber. It is clarified that its excellent viscoelastisity heavily owes to one of the main components, hyaluronic acid.
Concentration and molecular weight analyses of hyaluronic acid demonstrated the concentration and molecular weight of hyaluronic acid in the synovial fluid from patients with arthritis such as osteoarthritis and chronic articular rheumatism generally tend to lower than in normal synovial fluid, and the lower concentration and molecular weight of hyaluronic acid are closely associated with development of locomotor dysfunction and pain attributable to the weaker lubricating action and the weaker protecting action on the surface of the articular cartilage of synovial fluid.
Injection of high molecular weight hyaluronic acid solution (Artz: from Seikagaku Corporation, average molecular weight 900000; Hyalgan: from Fidia, average molecular weight<500000) into diseased joints has been widely adopted as an effective measure for osteoarthritis among those articular diseases, and the source of high purity hyaluronic acid preparations for this purpose is cockscombs.
Such hyaluronic acid preparations from cockscombs are biologically inherent and quite safe but usually have to be administered as frequently as several to 10 times to show significant therapeutic effect.
Persistency tests on rabbits revealed that hyaluronic acid with a molecular weight of less than 1000000 administered into the knee joint cavities disappears from the knee joint cavities in 1 to 3 days and suggest the need of frequent administrations (Blood Coagulation and Fibrinolysis, vol 12, 173, 1992).
On the other hand, the molecular weight of hyaluronic acid found in the living body is reported to be as high as millions to 10000000, and a crosslinked hyaluronic acid derivative [Hylan: from Biomatrix] obtained by treatment with a chemical crosslinker has been developed as a therapeutic agent for knee joints with the idea that high molecular weight hyaluronic acid closer to the biologically intact one is likely to have higher effect.
Reportedly, the crosslinked hyaluronic acid persisted for a period as long as 20 to 30 days after administration into rabbit knee joint cavities in the above-mentioned persistency tests and produced sufficient effect when administered three times in clinical tests, and is practically used as a therapeutic agent for arthritis (Journal of Rheumatology vol. 20, 16, 1993).
(II) Next, concerning emboli, treatments through embolization are known to effective for various diseases such as angiopathy, paraplastic aneurysm and varix. Obstruction of arteries as the nourishing channels for tumours is also effective in tumour treatment.
Some proposals have been made for embolization. For example, a balloon embolization method using a balloon-tip catheter has been developed (W. Taki et al., Surg. Neurol, Vol. 12, 363, 1979). In addition, a method in which 2-hydroxyethyl methacrylate (HEMA) is introduced into a balloon together with a polymerization catalyst through a catheter is also known (W. Taki et al., Surg. Neurol, Vol. 13, 140, 1980).
For cancer treatment through embolization, use of cisplatin-containing chitin (Tahara et al., Cancer and Chemotherapy, vol. 21(13), 2225, 1994), use of poly(benzyl 1-glutamate) microspheres carrying cisplatin (Li C et al., Parm, Res., Vol. 11(12), 1792, 1994) and use of SMANCS and Lipiodol suspension together with gelatin sponge as a embolizing material (Nakamura et al., Cancer and Chemotherapy, vol. 22(11), 1390, 1996) have been reported. In addition, poly(DL-lactate) microspheres are reported as a suitable material for use in embolismic chemotherapy in combination with continuous injection of a chemotherapeutic agent (Flandroy P et al., J Control Release, Vol. 44(2/3), 153, 1997) while it is mentioned that they have to biodegrade in a couple of days so that when this therapy is practiced repeatedly.
There are a lot of problems such as the short time obstruction in the balloon embolization due to shriveling of the balloon as a bar to production of satisfactory effect and the possibility of polymerization of monomers such as HEMA inside the catheter. Most embolizing materials used in embolismic chemotherapy are synthetically available and hardly biodegradable and doubtful in respect of biocompatibility. Poly(DL-lactate) microspheres, though biodegradable, do not guarantee complete safety when repeatedly administered.
Though highly biocompatible hyaluronic acid has no problem with safety, hyaluronic acid does not embolize when merely administered in the form of solution, and is required to have improved local persistency.
(III) Concerning soft tissues, the idea of injecting various materials to repair or swell soft tissues has rapidly developed since the invention of the subcutaneous injection needle, and a number of materials have been injected into human bodies to remedy soft tissues and skins. Among them, liquid silicone has been used widely for injection but is not used as much recently as it used to be due to its side effects such as skin ulceration attributable to its long retention time. Collagen has also been injected so far in various forms such as chemically crosslinked forms and fibrous forms. Crosslinked solid collagen requires incision to be injected and has problems in plasticity and flexibility. There is a disclosure about fibrous collagen in U.S. Pat. No. 3,949,073.
However, it shrinks in volume as its liquid components are absorbed and has to be supplemented. Injectable types of collagen like this can hardly be freed of contaminants such as immunity substances, are costly and do not necessarily have appropriate physical properties.
Hyaluronic acid has also been attempted as an injection for soft tissues (Ann. Plast. Surg., Vol. 38, 308, 1997). Because hyaluronic acid in solution is rapidly absorbed in vivo, various methods for chemical crosslinking of hyaluronic acid have been attempted to improve persistency and retention in soft tissues (U.S. Pat. No. 4,582,865, JP-B-6-37575, JP-A-7-97401, JP-A-60-130601).
And hylan B gel is commercially available as Hylaform in Europe (The Chemistry Biology and Medical Application of Hyaluronan and its Derivatives Vol. 72, p 278, PORTLAND PRESS).
(IV) Next, reference will be made to the posterior part of the eyeball, especially the retina bordered on the vitreous body. The retina marks the posterior boundary of the intraocular space, while the lens and the ciliary body mark the anterior boundary. The retina consists of two layers, the receptor layer of photosensitive cells in contact with the vitreous humor and the layer of pigment epithelial cells adjacent to the choroid. Liquid infusion into the receptor layer causes retinal detachment, separating the two layers of the retina.
For treatment of retinal detachment, the peeled retina is brought into contact with the pigmented epithelial layer and fastened by photocoagulation or cryocoagulation. The contact is achieved by pressing an inward buckle against the sclera and the choroids from outside or by generating pressure from vitreous humor onto the retina through volume expansion of vitreous humor by injection.
In the latter case, when vitreous humor has to be removed partly or completely due to too much spilt blood for reabsorption or inward growth of the retina accompanying retinal detachment, various materials have been attempted as artificial vitreous bodies.
These artificial vitreous bodies are intended to maintain the shape of the eyeball and bring back the retina in position by pressing the retina against the pigmented epithelium in the vitreous chamber for a while.
As artificial vitreous bodies, physiological saline, glycerin, animal vitreous bodies, air, various gases, polyvinyl alcohol, collagen gel, silicone oil, hyaluronic acid and perfluorocarbons may be mentioned, and air, gases such as sulfur hexafluoride, silicone oil, liquid perfluorocarbons such as perfluorooctane and perfluorodecalin are generally used now.
Various expansive gases are used as artificial vitreous bodies by themselves or in mixtures with air, and have proven to be useful (American Journal of Opht hyaluronic acid lmology, Vol. 98, 180, 1984).
However, they sometimes cause complications such as increase of intraocular pressure and coreclisis attributable to gas expansion or keratoleukoma attributable to their contact with the corneal endothelium and impose on patients a heavy burden of keeping their faces down for a long time.
Silicone oil maintains the intraocular space for a longer time than gases by virtue of its little absorbability and accelerates adhesion of the retina effectively (Retina, Vol. 7, 180, 1987), but is used with the proviso that it is drawn out after exertion of the pressing effect on the retina. Further, it is said to have serious problems of cataract, glaucoma and toxic effects on the ocular tissue (Ophthalmology, Vol. 27, 1081, 1985).
Liquid perfluorocarbons as artificial vitreous bodies are proved to cause complications such as proliferative vitreoretinopathy, cataract and intraocular hypotension and are reported to be more questionable than silicone oil and gases in respect of safety and effectiveness (New Ophthalmology, Vol. 12, 1053, 1995).
Hyaluronic acid has been investigated a lot since Balazs reported its application in the field of ophthalmology (Mod. Probl. Opht hyaluronic acid lmol., Vol. 10, 3 1972) and is widely used in ophthalmic surgery, especially intraocular implantation.
Hyaluronic acid is inherently biogenic and never induces toxic or immunological reactions. However, hyaluronic acid can not exert the effect of maintaining the intraocular space for a long time sufficient for treatment of serious retinal detachment because hyaluronic acid injected into the vitreous chamber dissolves in aqueous humor and is discharged from the eye through the anterior chamber and the fibrous trabecular goniomeshwork without being decomposed.
Though as vitreous injections containing hyaluronic acid, for example, those containing at least 1.5 wt %, preferably from 2 to 2.5 wt % of hyaluronic acid with a molecular weight of at least 900000, preferably 1600000 to 2000000 are disclosed in JP-A-5-184663, they are not retained in the intraocular space [Nippon Ganka Kiyou, vol. 38, 927, 1987], and over 1.5 wt % solution of hyaluronic acid with such a molecular weight strains a syringe when ejected from the syringe into the vitreous body and is not practical.
As mentioned above, improvement of the in vivo retention of hyaluronic acid is essential for its applications, and various chemical crosslinkers have been used to crosslink hyaluronic acid (U.S. Pat. No. 4,582,865, JP-A-60-130601, JP-A-63-281660, JP-B-6-37575, JP-B-6-69481, JP-A-7-97401, JP-A-7-59303). Further, production of a photo-crosslinked hyaluronic acid gel by irradiation of a photo-crosslinkable hyaluronic acid derivative with ultraviolet light is also known (JP-A-143604).
However, these cross-linked products of hyaluronic acid are not what is called hyaluronic acid any longer, and among the desired properties for materials used in vivo, non-toxicity and non-immunogenicity can not absolutely be secured for them considering procedures for removal of crosslinkers and the difficulty of complete denial of the presence of residual crosslinkers.
The present inventors have found out a simple method of producing a hardly water soluble hyaluronic acid gel made of hyaluronic acid alone for the first time (PCT/JP98/03536). However, the gel is sheet-like, filmy, flaky, spongy or massive and lacks transparency.
Therefore, the present inventors conducted extensive research with an idea that a hardly water soluble transparent material containing hyaluronic acid would be useful and find various medical applications.
To take the advantages of the excellent biocompatibility hyaluronic acid inherently has by itself to the maximum, hardly water soluble hyaluronic acid gels with transparency obtainable without using any chemical crosslinker or modifier are favorable. But such gels had not been developed yet.
On the other hand, for use of hyaluronic acid gels in the field of ophthalmology, especially as artificial vitreous bodies, transparency is required in view of effectiveness. Further, gels with refractive indices closer to that of the vitreous body are preferable (1.3345–1.3348; (Ganka Shinryo Practice, Vol. 22, pp 234, 1996, Bunkodo, Tokyo). However, no gels have been developed yet that have these properties.
The present inventors thought that impartment of transparency to a hardly water soluble hyaluronic acid gel made of hyaluronic acid alone obtained without using any crosslinker or the like would broaden the applications of hyaluronic acid gels and as a result of extensive research for such a gel, have found that hyaluronic acid forms a hyaluronic acid gel when kept in water at a hyaluronic acid concentration of at least 5 wt % in the presence of an acid component in an amount at least equimolar with the carboxylic groups in the hyaluronic acid and that the hyaluronic acid gel obtained in accordance with the present invention is characterized by transparency.